And High-Dose Ethanol Intake on the Nitrergic Relaxations of Corpus Cavernosum and Penile Nitric Oxide Synthase in the Rabbit

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ORIGINAL ARTICLE Effects of chronic low- and high-dose ethanol intake on the nitrergic relaxations of corpus cavernosum and penile nitric oxide synthase in the rabbit

Y Yazir1, SS Gocmez2, T Utkan3, I Komsuoglu-Celikyurt4, N Gacar4 and Y Sarioglu5

Epidemiological evidence showed that chronic ethanol consumption is a major risk factor in the development of impotence. The present study investigated the effects of carbachol-, electrical ﬁeld stimulation (EFS)-, sodium nitroprusside (SNP)- and papaverine-induced relaxant responses in the isolated corpus cavernosum tissues from rabbits submitted to an 12-week course of chronic low (5% v/v) or high ethanol intake (30% v/v). Increased carbachol- and EFS-induced relaxant responses but not SNP and papaverine, were observed in low ethanol-fed rabbits compared with controls. However, impaired carbachol- and EFS- induced relaxant responses were observed in high ethanol-fed rabbits compared with control rabbits. There were no signiﬁcant differences in SNP- and papaverine-induced relaxant responses between control and high ethanol-fed rabbits. In addition, decreased neuronal nitric oxide synthase (nNOS) and endothelial NOS (eNOS) immunoreactivity in penile tissue were found in high ethanol-fed rabbits, but increased the immunoreactivity in low ethanol-fed group, compared with control group. These results suggest that alterations in nitric oxide (NO) production within the cavernous tissue in the high ethanol-fed rabbits are, at least in part, responsible for the erectile dysfunction.

International Journal of Impotence Research (2012) 24, 185--190; doi:10.1038/ijir.2012.14; published online 10 May 2012 Keywords: alcohol consumption; carbachol; corpus cavernosum; impotence; nitric oxide

INTRODUCTION ethanol intake on the nitrergic-mediated relaxant responses of the Many epidemiological studies have shown that chronic ethanol rabbit corpus cavernosum and to determine penile endothelial consumption is associated with increased incidence of impotence.1 nitric oxide synthase (eNOS) and neuronal NOS (nNOS) expression. Generally, low levels of alcohol ingestion increase male sexual To our knowledge, there are no similar studies examining the activity by increasing libido; however, higher alcohol levels impair effect of chronic high dose of ethanol on the nitrergic relaxation of the penile tumescence and reduce sexual performance.2 Nitric corpus cavernosum smooth muscle. oxide (NO) is possibly the principal neurotransmitter for cavernous smooth muscle relaxation, the key step in the erectile process.3 In MATERIALS AND METHODS recent years, impaired NO-mediated relaxant responses of corporal tissue has been recognized as an important cause of impotence in Experiments were performed on mature male New Zealand White rabbits various pathological conditions such as diabetes mellitus,4 aging,5 (2--2.5 kg) obtained from the Experimental Medical Research and Applica- hyperthyroidism,6 pudental arterial occlusion,7 hypercolesterole- tion Center (DETAB, Kocaeli University, Kocaeli, Turkey). The rabbit was mia,8 cavernous nerve denervation9 and smoking.10 Although chosen as the model based on the close similarities that have been chronic alcohol consumption results in impotence, Saito et al.11 reported in the reactivity in vitro of human and rabbit corpus cavernosum. demonstrated that in vivo chronic ethanol administration (5% v/v) Ethical approval was granted by the Kocaeli University Animal Research augmented acetylcholine- and electrical ﬁeld stimulation (EFS)- Ethics Committee (Kocaeli, Turkey). induced relaxant responses of corporal smooth muscle. Also, many reports in the literature revealed that ethanol increases the Treatment schedule synthesis or release of NO in isolated arteries and cultured The rabbits were divided into three groups: 9 rabbits in group 1 received endothelial cells.12,13 On the other hand, some investigators noted tap water ad libitum, and 9 each in groups 2 and 3 were fed 5% ethanol that ethanol decreased endothelium-dependent relaxations in and 30% ethanol, respectively. The model of ethanol feeding was that isolated arteries.14--16 Recent studies on experimental animals and described previously in which rabbits received 5% ethanol (vol/vol) in the humans indicated that the effects of ethanol on NO availability are drinking water for the ﬁrst week, 10% for the next 2 weeks, 20% from highly dependent on whether it is taken at low or high weeks 4--6 and 30% from weeks 6--12 in the group 3.18 In the group 2, concentration and acute or chronic intake.17 Therefore, the aim rabbits were received 5% ethanol constantly for 12 weeks. All rabbits had of the present study was to investigate the effects of chronic constant access to standard laboratory rabbit chow.

1Department of Histology and Embryology, Faculty of Medicine, Kocaeli University, Kocaeli, Turkey; 2Department of Pharmacology, Faculty of Medicine, Namik Kemal University, Tekirdaq, Turkey; 3Department of Pharmacology and Experimental Medical Research and Application Center, Faculty of Medicine, Kocaeli University, Kocaeli, Turkey; 4Department of Pharmacology, Faculty of Medicine, Kocaeli University, Koceli, Turkey and 5Department of Pharmacology, Faculty of Medicine, Gazi University, Ankara, Turkey. Correspondence: Professor Dr T Utkan, Department of Pharmacology and Experimental Medical Research and Application Center, Faculty of Medicine, Kocaeli University, 41380, Umuttepe, Kocaeli, Turkey. E-mail: [email protected] Received 14 June 2011; revised 5 October 2011; accepted 11 April 2012; published online 10 May 2012 Corporal relaxations in ethanol-fed rabbits Y Yazir et al 186 Strip preparation and organ bath studies (Invitrogen, CA, USA) on glass slides. Slides were examined under light The rabbits from each group were anesthetized with ketamine (25 mg/kg, microscope (Olympus BX 50, Tokyo, Japan) and photomicrographs were i.p.) and xylazine (5 mg/kg, i.p.) and exsanguinated 12 weeks post taken with Leica DM 100 (Leica DFC 290HD, Wetzlar, Hessen, Germany). All treatment, as peviously described.19 Brieﬂy, the penis was dissected free samples were treated with exactly the same protocol. Two independent and removed at the level of the crural attachments to the puboischial observers graded the staining intensity on a semi-quantitative scale bones. The specimen was immediately placed in Kreb’s bicarbonate buffer ranging from no expression (À), very weak ( þ ), moderate ( þþ), strong

composed of (mM): NaCl 118, KCl 4.7, CaCl2 2.5, NaHCO3 25, MgSO4 1.2, ( þþþ) to very strong ( þþþþ) expression. KH2PO4 1.2 and glucose 11. The tunica albuginea was cleared of the overlying tissue and opened. The proximal half of the corporal body was Biochemical parameters dissected free from the tunica and harvested en bloc as previously 5,7,9 Blood glucose, triglyceride, total cholesterol and high-density lipoproteins described. Each corporal body was cut transversely to obtain two (HDL) levels were measured in the rabbit’s blood using Abbott Architect longitudinal strips. Each strip was mounted in a 20-ml organ bath c16000 autoanalyzer (Abbott Laboratories, IL, USA). Also testosterone containing Kreb’s bicarbonate buffer equilibrated with a gas mixture of levels were measured in blood using Roche Analytics E170 Immnunology 1 95% O2 and 5% CO2 and maintained at 37 C. Recording of isometric strip Analyzer (Roche, Tokyo, Japan). tension were made using a transducer (MAY-COM, FDT 10 A, COMMAT Iletisim, Ankara, Turkey) and recorded online on a computer via a four channel transducer data acquisition system (TDA 94, COMMAT ILETI˙SI˙M, Analysis of data Turkey) using appropriate software (Polywin 95 1.0 COMMAT ILETI˙SI˙M, Experimental values are expressed as the mean±s.e.m. The relaxant Turkey). The resting load was set at 2 g, a value that has been previously effects of the agonists are expressed as percantage of the precontraction found to be optimal for the measurement of changes in the tension response to phenylephrine. To evaluate the effects of the agonists, pD2 of rabbit corpus cavernosal tissue preparations.5,7,9 The preparations (that is, the negative logarithm of the concentration for the half-maximal were allowed to equilibrate in Kreb’s bicarbonate buffer for 1 h and response; EC50) and maximum response (Emax) values were calculated. during this time Kreb’s bicarbonate buffer was replaced every 15 min with Agonist pD2 value was calculated from each agonist dose--response curve fresh solution. by linear portion of the curve and taken as a measure of the sensitivity of The corpus cavernosum strips in organ chambers were contracted with the tissues to each agonist. À6 À8 À4 10 M phenylephrine and added to carbachol (10 --10 M), sodium Statistical comparison between the groups was performed using analysis À8 À4 À5 À4 nitroprusside (SNP; 10 --10 M) and papaverine (10 --10 M). Trans- of variance followed by Tukey’s test. The score of immunoreactivity was mural EFS was provided by a stimulator (ST95 PT, COMMAT ILETISIM, assessed by the Kruskal--Wallis Test. Probabilities of Po0.05 were con- Turkey) and applied via two platinum wire electrodes set vertically within sidered significant. the organ bath on the opposite sides of the suspended tissue. Before electrical stimulation, the tissue was treated with guanethidine (5 mM; an Drugs adrenergic nerve blocker) and atropine (1 mM; a muscarinic receptor The following drugs were all obtained from Sigma Chemical (St. Louis, MO, blocker) for 30 min. Strips were precontracted with phenylephrine at USA): carbachol chloride, phenylephrine hydrochloride, SNP, atropine sulfate, À5 10 M, EFS was performed. Square-wave pulses of 10 V with a 0.5-ms guanethidine sulfate and papaverine hydrochloride. All drugs dissolved in duration in 10-s trains with varying frequencies (2--32 Hz) were applied at distilled water and were freshly prepared on the day of the experiment. 5-min intervals. The strips were allowed to return to baseline precontractile tension at each frequency. RESULTS Immunohistochemistry The contractions elicited by KCl (124 mM) were similar in all the three groups (Table 1). Cavernous specimens were fixed in 10% neutral-buffered formalin. The specimens were embedded in regular paraffin wax and cut into 3-mm thick sections. Tissue sections were deparaffinized in xylene and Effects of low- and high-dose ethanol consumption on rehydrated in ethanol. Antigen retrieval was performed by microwave endothelium-dependent relaxation À8 À4 oven at 600W for 3 Â 5 min in the citrate buffer. Endogenous peroxidase Carbachol (10 --10 M) produced concentration-dependent À6 was blocked by 3% H2O2 in methanol 15 min and again washed three relaxation in submaximally precontracted (10 M phenylephrine) times in phosphate-buffered saline. Afterwards, sections were incubated in corpus cavernosum strips obtained from control, low-dose and a blocking serum (Histostatin plush kit broad spectrum, Invitrogen, CA, high-dose alcohol-fed rabbits. In the low-dose alcohol consump- USA) for 10 min at room temperature to block non-specific binding. tion group, the carbachol concentration--response curve was Subsequently, sections were incubated over night at room temperature

with primary goat anti-nNOS polyclonal antibody (abcam-ab1376) at 2 mg/ Table 1. Maximum responses (Emax) for carbachol, sodium ml and 20 min at room temperature with primary anti-eNOS polyclonal nitroprusside (SNP), papaverine and KCl in corpus cavernosum strips antibody (abcam-ab87750) at 5 mg/ml in a humidiﬁed chamber. Negative obtained from control and alcohol-fed groups control incubations were performed by replacing the primary antibody with the appropriate non-immune immunoglobulin G in the same Control 5% Alcohol-fed 30% Alcohol-fed concentrations. Sections were washed three times in phosphate-buffered saline and incubated with the biotinylated secondary antibodies (Histos- Carbachol (%) 67.9±4 90.4±4.2* 42.8±4.4* tatin plush kit broad spectrum, Invitrogen, CA, USA) for 20 min at room EFS (%) 52.1±8.5 83.7±5.1* 33.6±1.9* temperature. After three washes with phosphate-buffered saline, the SNP (%) 98.8±0.5 98.7±0.4 97.3±0.7 sections were incubated with peroxidase labeled streptavidin (Histostatin Papaverine (%) 98.6±1.4 97.5±3.4 98.0±2.7 ± ± ± plush kit broad spectrum, Invitrogen, CA, USA) for 10 min. Peroxidase KCl (mg) 3144.6 466.3 3352.6 402.6 3422.6 266.1 activity was visualized with 3-amino-9-ethylcarbazol chromogen in large- Abbreviations: EFS, electrical ﬁeld stimulation; KCl, potassium chloride. volume 3-amino-9-ethylcarbazol substrate (AEC red kit 00-2007, Invitrogen, *Po0.05, statistically different from the response from control rabbits CA, USA) for nNOS and with 3, 3’-diaminobenzidine substrate (DAB kit 88- (n ¼ 9 in each group). À6 2014, Invitrogen, CA, USA) for eNOS. All incubations were performed in a Emax (Percentage of 10 M phenylephrine contractile response) values for moist chamber at room temparature using phosphate-buffered saline for carbachol, EFS, SNP, papaverine and Emax (mg of contractile response) ± washes between incubation steps. The sections were counterstained with values for KCl. Values are arithmetic means s.e.m, n ¼ the number of preparations used. Mayer’s hematoxylin (Invitrogen, CA, USA) and mounted with Clearmount

International Journal of Impotence Research (2012), 185 -- 190 & 2012 Macmillan Publishers Limited Corporal relaxations in ethanol-fed rabbits Y Yazir et al 187 shifted to the left and Emax values were increased compared with Effects of low- and high-dose ethanol consumption on penile control rabbits (Po0.05; Figure 1, Table 1). There were no nNOS and eNOS expression signiﬁcant changes in pD2 values (Table 2). However, in the Immunopositive staining was found after nNOS or eNOS high-dose alcohol consumption group, the carbachol concentra- immunohistochemistry in penile tissue. Weak immunostaining tion--response curve was shifted to the right with signiﬁcantly was observed in penile tissue in high ethanol-fed group (Figures lower pD2 value and Emax values were decreased compared with control rabbits (Po0.05; Figure 1, Tables 1 and 2).

Table 2. pD2 values for carbachol and sodium nitroprusside (SNP) in Effects of low- and high-dose ethanol consumption on corpus cavernosum strips obtained from control and alcohol-fed endothelium-independent relaxation groups À10 À4 In precontracted strips, SNP (10 --10 M) and papaverine À5 À4 (10 --10 M) produced concentration-dependent relaxation. Control 5% Alcohol-fed 30% Alcohol-fed The relaxation elicited by both SNP and papaverine was similar Carbachol 5.92±0.24 5.79±0.24 4.82±0.28* in low-dose alcohol consumption, high-dose alcohol consumption SNP 5.50±0.17 5.45±0.18 5.60±0.08 and control groups, and there were no signiﬁcant changes in the pD2 and Emax values (Figure 2, Tables 1 and 2). *Po0.05, statistically different from the response from control rabbits (n ¼ 9 in each group). Values are arithmetic means±s.e.m. Effects of low- and high-dose ethanol consumption on neurogenic relaxation In precontracted strips, EFS (2--32 Hz) evoked frequency- dependent relaxations. In the low-dose alcohol-fed group, EFS 100 Control (n=9) responses were higher than those in the control group (Po0.05; 5% Alcohol (n=9) 30% Alcohol (n=9) Figure 3, Table 1). But in the high-dose alcohol-fed group, EFS 80 responses were inhibited compared with the control group (Po0.05; Figure 3, Table 1). 60

Blood glucose, triglyceride, total cholesterol, HDL and testosterone 40 levels in control and alcohol-fed rabbits % Relaxation Levels of blood glucose, triglyceride, total cholesterol, HDL and testosterone in control and alcohol-fed rabbits are shown in 20 Table 3. The blood glucose (mg/dl) and HDL levels (mg/dl) of 5% alcohol-fed and 30% alcohol-fed rabbits were similar to the 0 control group. Cholesterol (mg/dl) and triglyceride (mg/dl) levels 10-10 10-9 10-8 10-7 10-6 10-5 10-4 were signiﬁcantly higher in 30% alcohol-fed rabbits compared Sodium nitroprusside (M) with the control group (Po0.05). Additionally, testosterone level (ng/dl) was signiﬁcantly lower in 30% alcohol-fed rabbits Figure 2. Sodium nitroprusside concentration--response in isolated rabbit corpus corpus cavernosum strips precontracted with pheny- compared with that of the control group (Po0.05). À6 lephrine 10 M. Each point is expressed as a percentage of the contraction induced by phenylephrine and is given as the mean±s.e.m. Numbers in parentheses indicate the number of preparations used.

Control (n=9) 5% Alcohol (n=9) Control (n=9) 30% Alcohol (n=9) 100 100 * * 5% Alcohol (n=9) * * 30 % Alcohol (n=9) * * * * 80 * * * * * * * * 60 * 50 * * * 40 % Relaxation % Relaxation * * 20 * * * 0 0 10-8 10-7 10-6 10-5 10-4 0 8 16 24 32 Carbachol (M) EFS (Hz) Figure 1. Carbachol concentration--response in isolated rabbit Figure 3. Relaxation responses evoked by EFS of isolated rabbit À6 corpus corpus cavernosum strips precontracted with phenylephrine corpus cavernosum strips precontracted with phenylephrine 10 M. À6 10 M. Each point is expressed as a percentage of the contraction Each point is expressed as a percentage of the contraction induced induced by phenylephrine and is given as the mean±s.e.m. by phenylephrine and is given as the mean±s.e.m. Numbers in Numbers in parentheses indicate the number of preparations parentheses indicate the number of preparations used. *Po0.05, used. *Po0.05, statistically different from the response from control statistically different from the response from control rabbits. EFS, rabbits. electrical ﬁeld stimulation.

& 2012 Macmillan Publishers Limited International Journal of Impotence Research (2012), 185 -- 190 Corporal relaxations in ethanol-fed rabbits Y Yazir et al 188 4c and f) compared with control (Figures 4a and d; Po0.05), chronic alcohol-induced hypertension is associated with the indicating the NO generation through nNOS or eNOS is impaired downregulation of antioxidants and NO production in the aorta in the penile tissue of the high-dose ethanol-fed rabbits. Contra- of rats.14--16 rily, in low ethanol-fed group (Figures 4b and e), increased NO plays an essential role in the erection of penis. It is well immunostaining was observed compared with the control group known that both the autonomic nerves that innervate corpus (Figures 4a and d; Po0.05). cavernosum are independent sources of NO.27,28 In this study, EFS and exogenous application of SNP, papaverine and carbachol produced relaxation responses in both alcohol-treated and DISCUSSION control groups. There were no significant differences in response The present study addresses the changes in neurogenic and to SNP and papaverine in all the groups. However, the relaxation endothelium-dependent relaxation to carbachol in the corporal response to EFS and carbachol were significantly increased smooth muscle and its relationship with blood alcohol levels in in low-dose alcohol-treated rabbits. Therefore, it is speculated rabbit-ingested ethanol at different doses for 12 weeks. In most of that low-dose alcohol consumption increased production of the human studies, chronic alcohol consumption is a major risk NO. It is also possible that low-dose alcohol consumption may factor in the development of impotence. Also, it is well known that increase relaxation of trabecular smooth muscle or increase the concentration and duration of ethanol ingestion have an the ability to relax via the NO/cGMP pathway; however, this important impact on erectile outcomes in animal models. possibility is unlikely as the strips relaxed well to papaverine or Experiments on alcohol and erectile response showed that high SNP. Similarly, we have previously shown increased endothelium- dependent relaxant response to acetylcholine in isolated aortas doses of alcohol attenuate erectile response; however, light-to- 29 moderate alcohol consumption increases male sexual activity.20--22 from 8-week ethanol-treated (7.5%) rats. Our findings are The results obtained with the corpus cavernosum were consistent consistent with previous studies demonstrating that chronic moderate alcohol ingestion enhanced NO production by vascular with previous studies showing that light-to-moderate alcohol 13 consumption is inversely associated with cardiovascular dis- cells in vitro. Our evidence indicates that chronic light alcohol eases.23--25 It has been proposed that increased NO production ingestion increases trabecular NO production and causes is a major physiological system for the protective action of increased trabecular smooth muscle relaxation leading to increase moderate alcohol consumption.12,13 However, experimental and in sexual performance. epidemiological evidence show that chronic high-dose ethanol Also, in this study, EFS- and carbachol-induced relaxation consumption increases mortality and causes cardiovascular responses were significantly decreased in the higher-dose complications, including hypertension.26 It is hypothesized that alcohol-treated group. The results of the present study further showed that penile nNOS significantly decreased in high ethanol consumption, indicating erectile dysfunction. It is reported that, when rats treated with graded concentrations of alcohol for 12 Table 3. Blood glucose, triglyceride, total cholestrol, HDL and weeks, higher concentrations of alcohol were associated with testosterone levels in control and alcohol-fed rabbits hypertension and reduced plasma levels of NO metabolites. Authors recently clarified that daily oral treatment with ethanol for Control 5% Alcohol-fed 30% Alcohol-fed 12 weeks caused significant decreases in aortic NO production, reduced endothelium-dependent vasorelaxation and increased Glucose (mg/dl) 143.33±1.47 141.67±23.64 149±7.07 blood pressure that were due to, in part, alcohol-induced Cholesterol (mg/dl) 42±10.14 31.83±4.64 93.3±11.74* reductions in eNOS.14 This finding is consistent with our results, ± ± ± HDL (mg/dl) 11 3.24 17 4.15 19.83 5.05 therefore we suggest that impaired NO production have an Triglyceride (mg/dl) 57.83±8.31 74.5±8.50 181.55±21.30* Testosterone (ng/dl) 4.36±0.56 3.79±0.62 0.95±0.24* important pathophysiological role in the erectile dysfunction after high-dose alcohol consumption in rabbits. Abbreviation: HDL, high-density lipoprotein. In addition, several mechanisms have been proposed to *Po0.05, statistically different from the response from control rabbits account for the pathogenesis of impotence associated with (n ¼ 9 in each group). chronic alcohol consumption such as impairment of hypotalamic ± Values are arithmetic means s.e.m. function.30 Androgens are essential in the maintenance of libido

nNOS

eNOS

Figure 4. Representative light microscopy of control and chronic ethanol-fed groups in penile tissue. Decreased (c) neuronal nitric oxide synthase (nNOS) and (f) endothelial NOS (eNOS) immunoreactivity at corpus cavernosum in high-dose ethanol-fed rabbits and increased (b) nNOS and (e) eNOS immunoreactivity at penile tissue in low-dose ethanol-fed rabbits compared with (a, d) control groups.

International Journal of Impotence Research (2012), 185 -- 190 & 2012 Macmillan Publishers Limited Corporal relaxations in ethanol-fed rabbits Y Yazir et al 189 and have an important role in regulating erectile capacity in man. collection, analysis and interpretation of data, in the writing of the report and in the In hypogonadal patients, it is known that exogenous testosterone decision to submit the paper for publication. administration stimulates erectile function.31 Previous studies have shown that alcohol generally decreases circulating testosterone levels in animals and humans, but many man who are alcohol CONFLICT OF INTEREST dependent have normal testosterone and estrogen levels.32--34 The authors declare no conflict of interest. Cicero and Badger32 reported that low doses of ethanol significantly increased serum testosterone, whereas high doses REFERENCES decreased serum levels of testosterone, in male Spraque--Dawley rats. 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